Peptides and binding partners therefor

ABSTRACT

The invention provides a peptide obtainable from  C. albicans  as well as variants and fragments thereof, and labelled forms of these. The peptide is immunogenic and specific binding partners for the peptide and labelled forms of these specific binding partners form a further aspect of the invention. The peptide is a fragment of the ECE1 protein and has been found to be both immunogenic and act as a pore-forming toxin. A range of therapeutic and diagnostic applications for the peptide and the specific binding partners for it form further aspects of the invention. In addition, the peptide may be used in screens for identifying compounds having useful anti-fungal activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of U.S. patent application Ser.No. 14/783,237, filed Oct. 8, 2015, which is a National Stage ofInternational Patent Application No. PCT/GB2014/051118, filed Apr. 10,2014, which claims benefit of GB 1306588.3, filed Apr. 11, 2013, thedisclosure of each is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to moieties selected from a specific typeof peptide and binding partners such as antibodies therefor, which haveapplications in diagnosis and therapy. Methods for using these moietiesin diagnosis and therapy as well as in processes for screening fortherapeutic compounds are also part of the invention. The isolatedpeptide and specific binding partners therefor are novel and formfurther aspects of the invention.

BACKGROUND TO THE INVENTION

Candida species are the most common fungal pathogens of humans and thecausative agents of oral, gastrointestinal and vaginal candidiasis,giving rise to severe morbidity in millions of individuals worldwide.Vaginal candidiasis affects ˜75% of women at least once during fertileage, equating to −30 million infection episodes/year (3× more thantuberculosis and 8× more than HIV: WHO 2007). Candida infections arealso the 3^(rd) most common hospital-acquired bloodstream infection,making Candida species more medically-important than most bacterialinfections including Enterococci (E. coli) and Pseudomonas spp. Systemiccandidiasis is fatal (30-50% mortality) with 300,000 cases/year ofcandidaemia, equating to 100,000 deaths/year. Furthermore, Candidainfections are the most common oral manifestation of HIV infection, with50% of HIV+ patients and 90% AIDS patients suffering from oralcandidiasis. With ˜4 million cases of HIV/year, this equates to −2million oral candidiasis cases/year. Indeed, one of the biggest killersof the immunocompromised population is fungal infection. In the USA,yearly healthcare costs for fungal infections are $2.6 billion, of whichCandida infections account for $1.8 billion. EU healthcare costs areestimated to be similar. Therefore, Candida pathogens carry an immensehealth burden and represent a major socio-economic challenge forworldwide communities.

Candida albicans is a member of the normal human microbiome. Althoughtypically a commensal of the oral cavity, gastrointestinal andurinogenitary tracts, C. albicans is also an extremely frequent cause ofsuperficial infections such as vaginitis. Moreover, common iatrogenicprocedures, such as gastrointestinal surgery, implantation of a centralvenous catheter or antibiotic treatment are major risk factors fordisseminated candidiasis. This form of systemic candidiasis is now thethird most common cause of nosocomial bloodstream infections and themortality of severe sepsis caused by Candida species is over 50% in somepatient groups.

Identifying new epithelial and fungal targets that stimulate protectivehost immunity will ultimately have major implications for global health.

C. albicans virulence relies on a number of factors, includingmorphological plasticity, the expression of adhesins and invasins,robust stress responses, immune evasion, metabolic flexibility andnutrient acquisition. However, it remains unclear, how C. albicanscauses damage.

The mechanisms by which C. albicans damages host cells have beenconsidered to be multi-factorial, and presumed to rely on a combinationof adhesion, invasion, hyphal extension, turgor pressure and thesecretion of hydrolytic enzymes. Although toxin production by C.albicans has long been postulated and the culture supernatants of C.albicans hyphae shown to exhibit haemolytic activity, the mechanismunderlying C. albicans ability to lyse host cells has remained elusive.It is clear, however, that hyphae are essential for adhesion, invasionand damage. Thus, damage is caused by hyphae and/or a hyphal associatedfactor.

The gene ECE1 (extent of cell elongation 1) was first identified twodecades ago due to its heightened expression during hypha formation.However, deletion of ECE1 did not affect hypha formation in C. albicansand phenotypic differences between C. albicans wild type and ECE1deletion strains have not been reported. Despite this, ECE1 expressionhas been used as a marker for hypha formation in multiple independentstudies and ECE1 is one of the most strongly expressed genes duringhyphal formation. In fact, ECE1 is within a small group of core hyphalassociated genes. It has previously been reported that recombinant Ece1is processed by the protease, Kex2, at lysine/arginine residues.However, the function of Ece1 has never been shown.

Currently, the gold standard for treating fungal infections is the useof antifungal drugs, which predominantly target either the cell wall(echinocandins), cell membrane (polyenes) or ergosterol synthesis(azoles). However, the continuing increase in Candida infectionstogether with increasing drug resistance highlights the need to discovernew and better agents that target either (i) epithelial processes thatrecognise and normally restrict these potentially life-threateningpathogens to mucosal surfaces or (ii) fungal determinants that promoteinfection and/or mediate immune activation and protective immunity.Despite this, there are no vaccine candidates or immune-basedintervention strategies for combating Candida infections. Likewise,there are no commercial drugs that specifically target mucosal fungalinfections.

SUMMARY OF THE INVENTION

The applicants have found that Ece1 plays a key role in host cellactivation and damage, as demonstrated by the fact that deletion of ECE1renders C. albicans unable to damage or activate inflammatory responsesin human epithelial cells. Moreover, the applicants have demonstratedthat a single peptide product of proteolytic processing of Ece1 acts asa peptide toxin, in particular, a pore-forming toxin: the first suchdescribed in a human fungal pathogen. By targeting this single peptideit is possible to prevent both host damage (fungus driven) andmanipulate immunity to induce protection (host driven). To date, noother Candida protein exhibits these phenotypes and no other approachcan address both fungal and host aspects.

This gives rise to a number of diagnostic and therapeutic applicationsfor this peptide and also for specific binding partners for it, as wellas for the discovery and development of anti-fungal compounds thattarget this specific peptide.

According to a first aspect of the present invention there is providedan optionally labelled peptide comprising SEQ ID NO 1

(SEQ ID NO. 1) SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKRor a variant thereof, or an immunogenic fragment of either of these; ora labelled form thereof.

As used herein, the expression ‘variant’ refers to a peptide sequence inwhich the amino acid sequence differs from the sequence of SEQ ID NO 1in that one or more amino acids within the sequence are substituted forother amino acids. However, the variant produces a biological effectwhich is similar to that of SEQ ID NO 1. In particular, any variant willinteract with the surface receptor EGFR (epidermal growth factorreceptor) to give rise to activation of an immune response and/orimmunoglobulins and in particular antibodies that are produced inresponse to said variants will cross-react with SEQ ID NO 1.Alternatively or additionally, the variant will be damage inducing inhost cells, for example by acting as pore forming agents.

Amino acid substitutions may be regarded as “conservative” where anamino acid is replaced with a different amino acid in the same classwith broadly similar properties. Non-conservative substitutions arewhere amino acids are replaced with amino acids of a different type orclass.

Amino acid classes are defined as follows:

Class Amino acid examples Nonpolar: A, V, L, I, P, M, F, W Unchargedpolar: G, S, T, C, Y, N, Q Acidic: D, E Basic: K, R, H.

As is well known to those skilled in the art, altering the primarystructure of a peptide by a conservative substitution may notsignificantly alter the activity of that peptide because the side-chainof the amino acid which is inserted into the sequence may be able toform similar bonds and contacts as the side chain of the amino acidwhich has been substituted out. This is so even when the substitution isin a region which is critical in determining the peptide's conformation.

Non-conservative substitutions may also be possible provided that thesedo not interrupt the function of the peptide and in particular itsability to cross-react with immunoglobulins that react with SEQ ID NO 1.

Broadly speaking, fewer non-conservative substitutions will be possiblewithout altering the biological activity of the polypeptides.

Particular variants may include peptides encoded by orthologues orparalogues of the gene sequence that encodes the peptide of SEQ ID NO 1in C. albicans. Particular variant sequences are orthologues from C.dubliniensis and C. tropicalis as shown in FIG. 2 hereinafter.

In general, variants will have amino acid sequences that will be atleast 70%, for instance at least 71%, 75%, 79%, 81%, 84%, 87%, 90%, 93%or 96% identical to SEQ ID NO 1. Identity in this context may bedetermined using the BLASTP computer program with SEQ ID NO 1 as thebase sequence. The BLAST software is publicly available atblast.ncbi.nlm.nih.gov/Blast.cgi (accessible on 12 Mar. 2009).

Variants may also include addition sequences such as tag sequences thatmay be used for instance in facilitating purification of the peptide orin detection of it. Thus for instance, the variant may further comprisean affinity tag such as chitin binding protein (CBP), maltose bindingprotein (MBP), glutathione-S-transferase (GST), FLAG, myc, biotin or apoly(His) tag as are known in the art. In another embodiment, thevariant may comprise a fluorescent protein such as green fluorescentprotein (GFP).

When the peptide is for use in diagnostics or screening of anti-fungalproteins, it may be helpful to label the peptide, for example with achemical label such as a fluorescent or radiolabel. There are a widerange of such labels available commercially, but examples includefluorescein and derivatives such as fluorescein isothiocyanates (FITC),carboxyfluorescein succinimidyl esters, fluorescein dichlorotriazine(DTAF) and 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),rhodamine and derivatives such as carboxytetramethylrhodamine (TAMRA),tetramethylrhodamine (TMR) and its isothiocyanate derivative (TRITC),sulforhodamine, Texas Red, Rhodamine Red and the dyes available underthe trade name Alexa.

These labels may be incorporated into the peptide using conventionalmethods.

The term “fragment” as used herein refers to any portion of the aminoacid sequence of SEQ ID NO 1 which has biological function in commonwith SEQ ID NO 1. (e.g. damage-inducing, immunomodulatory or isimmunogenic, for instance an epitopic fragment) and which reacts withspecific binding partners for SEQ ID NO 1. Fragments may comprise morethan one portion from within the full-length protein, joined together.Portions will suitably comprise at least 5 and preferably at least 10consecutive amino acids from the basic sequence. Suitable fragments willinclude deletion mutants comprising up to 31 amino acids, for example atleast 7 amino acids, such as at least 10, for instance at least 15, suchas at least 20, more suitably at least 30 amino acids.

Notably, SEQ ID NO 1 includes two segments that contain sequences withhigh amyloidogenic potential (underlined), which may be involved in cellinteractions and activation due to their hydrophobic nature and abilityto potentially form α-helices. Particular variants or fragments of SEQID NO 1 will contain at least one of said sequences which arerepresented below as SEQ ID NO 2 and SEQ ID NO 3 respectively.

(SEQ ID NO. 2) IIGIIMGIL (SEQ ID NO. 3) QVIQIIMSIV

In a particular embodiment however, the peptide is a peptide of SEQ IDNO. 1. The peptide is in isolated or purified form and in particular isfree of at least some and preferably all other peptides obtainable byKex2 protease cleavage of the Ece1 protein of C. albicans. Although thepeptide may be isolated from C. albicans that has been subject to Kex2protease digestion, in a particular embodiment, the peptide is preparedsynthetically using conventional preparation methods.

As discussed above, peptides of the invention and in particular thepeptide of SEQ ID NO 1 has been found to be immunogenic. Thus in asecond aspect of the present invention there is provided a specificbinding partner for a peptide as described above; or a labelled formthereof. Particular specific binding partners are immunoglobulins, suchas an antibody or a binding fragment thereof. Examples of suitablebinding fragments of antibodies include Fab, Fab′, F(ab)2, F(ab′)2 andFV, VH and VK fragments.

Antibodies may be polyclonal or monoclonal but in a particularembodiment, the antibodies are monoclonal antibodies.

Antibodies can be prepared using conventional methods involvinginoculation of an animal such as mouse or guinea pig with a peptide ofthe first aspect of the invention and harvesting antibodies from theblood thereof. Monoclonal antibodies can be obtained therefrom by fusingantibody producing cells such as spleen cells from the inoculated animalwith a hybridoma cell, culturing this cell and harvesting monoclonalantibodies therefrom.

Where the antibody is used in diagnostic or screening applications, itmay be chemically labelled to facilitate detection. Suitable labels willbe similar to those described above for the peptides of the invention.However, the antibody may be detectable using a secondary antibody whichis modified so as to be detectable using a development reaction, forexample because it includes an enzymatic label, as is conventional inthe art.

The peptide of the invention has therapeutic applications. Therefore, athird aspect of the invention provides a peptide as described above foruse in therapy, and in particular for the treatment or prevention ofinfection by Candida albicans. In particular, the therapy is for thetreatment or prevention of oral, gastrointestinal or mucosal and inparticular vaginal infection by Candida albicans.

In order to achieve these therapeutic effects, a non-toxic amount of apeptide as described above or a pharmaceutically acceptable compositioncomprising it is administered to a subject, such as a human or animal.The non-toxic amount is sufficient to produce an immune response that isprotective against C. albicans infection. Thus the peptide may be usedas a vaccine in a vaccination method. Such a method forms a fourthaspect of the invention.

The amount of peptide administered will vary depending upon factors suchas the size and health of the patient, the nature of the condition beingtreated etc. in accordance with normal clinical practice. Typically, adosage in the range of from 1 μg-50 mg/Kg such as from 1-50 μg/Kg but inparticular from 1-50 mg/Kg, for instance from 2-20 mg/Kg, such as from5-15 mg/Kg would be expected to produce a suitable immune response.Since the peptide of SEQ ID NO 1 has been found to have cytotoxiceffects however, care must be taken to ensure that the dosage issufficient to prime the immune system but not cause unwanted toxic sideeffects.

One way of avoiding such side effects is to use a variant or fragment ofthe peptide of SEQ ID NO 1 in the fourth aspect of the invention. Asdescribed hereinbelow, the applicants have found that full lengthpeptide of SEQ ID NO 1 is capable of inducing cellular lysis andstimulation of the inflammatory response in epithelial cells. These sideeffects may therefore be avoided by using a variant sequence which isimmunogenic, or a fragment of SEQ ID NO 1, in particular one whichcontains at least SEQ ID NO 2 or SEQ ID NO 3 as described above.Furthermore, the applicants have found that the peptide of SEQ ID NO 1interacts with phospholipids, omission of the dibasic (lysine, arginine)C-terminal head of the peptide may mitigate any unwanted toxic effects.

An alternative way of avoiding unwanted side effects is to use thespecific binding partners and in particular the antibodies of the secondaspect of the invention in a passive immunisation regime. Thus in afifth aspect of the invention, there is provided a specific bindingpartner for a peptide as described above for use in therapy, inparticular where the therapy is the treatment or prevention of infectionby Candida albicans.

As before, in order to achieve these effects, an effective amount of thespecific binding partner and in particular the antibody, or apharmaceutical composition containing it is administered to a subject inneed thereof. Thus the specific binding partner may be used as a vaccinein a vaccination method that may be prophylactic or therapeutic. Such amethod forms a sixth aspect of the invention.

Again, the amount of specific binding partner administered will varydepending upon factors such as the size and health of the patient, thenature of the condition being treated etc. in accordance with normalclinical practice. Typically, a dosage in the range of from 1 to 50mg/Kg, for instance from 2-30 mg/Kg such as from 5-10 mg/Kg wouldproduce a suitable therapeutic or protective effect.

For administration to subjects, the peptide or its specific bindingpartner is suitably administered in the form of a pharmaceuticalcomposition. Thus a seventh aspect of the invention provides apharmaceutical composition comprising a peptide as described above and apharmaceutically acceptable carrier.

An eighth aspect of the invention provides a pharmaceutical compositioncomprising a specific binding partner for a peptide as described aboveand a pharmaceutically acceptable carrier.

Suitable pharmaceutical compositions will be in either solid or liquidform. They may be adapted for administration by any convenient route,such as parenteral, oral, vaginal or topical administration or foradministration by inhalation or insufflation. The pharmaceuticalacceptable carrier may include diluents or excipients which arephysiologically tolerable and compatible with the active ingredient.

Parenteral compositions are prepared for injection, for example eithersubcutaneously or intravenously. They may be liquid solutions orsuspensions, or they may be in the form of a solid that is suitable forsolution in, or suspension in, liquid prior to injection. Suitablediluents and excipients are, for example, water, saline, dextrose,glycerol, or the like, and combinations thereof. In addition, if desiredthe compositions may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, stabilizing or pH-buffering agents,and the like.

Oral formulations will be in the form of solids or liquids, and may besolutions, syrups, suspensions, tablets, pills, capsules,sustained-release formulations, or powders. Oral formulations includesuch normally employed excipients as, for example, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,cellulose, magnesium carbonate, and the like.

Topical formulations will generally take the form of suppositories orintranasal aerosols. For suppositories, traditional binders andexcipients may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient.

Peptides of the first aspect and specific binding partners of the secondaspect of the invention, may, if required, be produced using recombinantmethods. For this purpose, nucleic acids that encode either the peptideor specific binding partner are prepared and these form a ninth aspectof the invention.

Such nucleic acids may be incorporated into a suitable expression vectoror plasmid, which is then used to transform a cell and in particular aprokaryotic cell. A suitable nucleic acid that encodes the peptide ofSEQ ID NO 1 may correspond to a nucleic acid sequence that occursnaturally in strains of C. albicans strains. However, in a particularembodiment, the nucleic acid is designed so that it is ‘codon optimised’for the expression host being used in the recombinant production method.This should ensure efficient and effective production. The design andpreparation of such nucleic acids will be apparent to the skilled personusing techniques conventional in the art.

A recombinant cell that has been transformed with such a nucleic acidforms a tenth aspect of the invention.

Suitable cells will be conventional expression hosts such as E. coli andPichia pastoris An eleventh aspect of the invention provides a methodfor producing a peptide as described above, or a specific bindingpartner therefor, which method comprises culturing a recombinant cell asdescribed above and recovering said peptide or specific binding partnertherefrom.

However, the peptides of the first aspect and the specific bindingpartners of the second aspect of the invention may also be useful indiagnosis of pathogenic invasive C. albicans infection. C. albicansforms part of the commensal flora and so may be found in mostindividuals at low levels, for example of up to 800 organisms per mlsample. However, as discussed previously, invasive or pathogenicinfections may arise, in particular in immunocompromised individuals,and in such cases, the level of C. albicans increases dramatically orthere is ‘overgrowth’. Although in many cases, pathogenic invasive oralor vaginal infection of C. albicans produces visible symptoms, it wouldbe helpful if tests could be provided that allow for early diagnosis ofsuch infection as this will allow treatment to be started before theinfection escalates to a more dangerous systemic infection, or for thediagnosis of invasive pathogenic infection in organs which may not beexamined easily such as the intestine.

In an eleventh aspect of the invention, there is provided a method fordiagnosis of pathogenic invasive infection by C. albicans, said methodcomprising contacting a sample from an individual with a peptide asdescribed above or a specific binding partner therefor, and detectingwhether the sample contains moieties that interact with said peptide orspecific binding partner at a sufficient level to indicate a pathogenicinvasive infection.

Detection of high levels of the peptide of the invention and inparticular a peptide of SEQ ID NO 1 in a sample may be indicative of anovergrowth of C. albicans in the individual, leading to the symptoms ofcandidiasis. This may be determined by using a specific binding partnerfor the peptide to capture it. The specific binding partner is suitablyeither directly or indirectly labelled so as to detect or visualise thepeptide in a quantitative or semi-quantitative manner.

Similarly, an elevated level of specific binding partner, and inparticular, antibodies to the peptide of SEQ ID NO 1 present in thesample can also indicate that an overgrowth of Candida has occurred andthat pathogenic invasive infection may result. In this case, thediagnostic method will typically utilise a peptide of the invention tobind with antibodies in the sample. The peptide may be immobilised orlabelled to assist in the capture and/or detection of the antibodies inthe sample.

The sample used in the method may be any suitable biological sampleincluding a blood, serum, sputum, saliva or mucosal swabs for instanceoral or vaginal swabs, but may also include biopsy samples such asintestinal tissue samples.

The levels of peptide or specific binding partner that is indicative ofa pathogenic invasive infection or Candida ‘overgrowth’ rather than thepresence of a non-damaging commensal species will vary depending uponfactors such as the nature of the sample and the particular patientbeing examined and the particular technique used to carry out the assay,but will generally be determinable in a particular case in accordancewith conventional clinical techniques. Typically however, levels of C.albicans of up to about 800 cells/ml saliva sample may be acceptable asa commensal background, but levels significantly higher than this, inparticular at least an order of magnitude greater than this, can beindicative of overgrowth and pathogenic invasive infection. Detection oflevels of the peptide of the invention at levels that equate to thiscellular population of Candida species would be a cause for concern.

Methods include those conventionally known in the art such asimmunoassays, for instance, enzyme-linked immuno sorbent assays (ELISA)but may also include multiplex bead assays, immunohistochemistry orimmunofluorescence. Such methods are well known in the art.

Kits for carrying out such methods form a further aspect of theinvention. Thus the invention further provides a kit for diagnosing apathogenic invasive infection by C. albicans, said kit comprising apeptide as described above or a specific binding partner therefor, andmeans for detecting complexes formed between said peptide or specificbinding partner and moieties in the sample. The nature of the detectionmeans will vary depending upon the particular technique being employed.In general, detection means will include label means. The label means issuitably one in which the intensity of the signal developed can berelated to the concentration of the moiety being detected as this willallow a distinction to be made between levels of C. albicans which iscommensal and a level at which disease may result.

Suitable label means may be indirect label means which allow signals todevelop following a subsequent reaction such as a chemical reaction.These will include enzymatic labels such as peroxidase labels and inparticular horseradish peroxidase. Alternatively, labels may includedirect labels such as visible plastic labels, fluorescent labels likefluorescein or derivatives thereof, rhodamine or derivatives asdescribed above, as well as radiolabels such as ¹²⁵I labels. The labelsmay be bound to or otherwise associated with the peptide of theinvention or the specific binding partner therefor, or they may belinked to a secondary detection moiety such as a secondary antibody orother binding moiety. The precise form the detection means will takewill vary depending upon the detection technique employed.

For instance for immunoassays such as ELISA assays, detection means mayinclude solid supports such as 96-well plates or beads that may beferromagnetic in nature. In such cases, the solid supports willgenerally have binding moieties for either the peptide of the firstaspect or the specific binding partner of the second aspect immobilisedthereon.

Where the binding moieties are specific for the peptide of theinvention, for instance they are specific binding partners of the secondaspect of the invention, incubation of the sample in contact with thesolid support will result in capture of the peptides on the support.Once the sample has then been removed from the support for example bywashing, the presence of immobilised peptide can be detected byapplication of a secondary detection antibody that also binds thepeptide but carries a label which is either directly or indirectlydetectable. Directly detectable labels may comprise visible labels suchas fluorescent or radiolabels as described above. Indirect labels may bedetected in subsequent reactions such as horseradish peroxidase that iscarried by the secondary detection antibody or by a tertiary detectionantibody that binds to the immobilised secondary antibody.

Similarly where the binding moieties on the solid support specific for aspecific binding partner and in particular an antibody to the peptide ofthe invention, they will generally comprising a peptide of the firstaspect of the invention. In this case, incubation of the sample incontact with the solid support will result in capture of antibodies tothe peptide on the support. Once the sample has then been removed fromthe support for example by washing, the presence of immobilised antibodycan similarly be detected by application of a secondary detectionantibody that binds the antibody but carries a label which is eitherdirectly or indirectly detectable.

For immunohistochemical or immunofluorescence detection methods, kitswill suitably contain labelled reagents such as suitable stains or dyesthat are targeted to the required moiety and in particular the peptideof the invention, as a result of attachment to a suitable specificbinding partner. In use, the sample will be prepared in the usual way.In particular, a tissue sample is collected, if necessary sectioned andfixed onto a support such as microscope slide whereupon the labelledreagent is administered to the slide so as to visualise the targetmoiety and in particular the peptide of SEQ ID NO 1. The kit maycomprise one or more additional elements used in immunohistochemical orimmunofluorescence method. These include the slide itself, fixing agentsuch as paraformaldehyde, detergent such as Triton® detergent forreducing surface tension and facilitating coverage by the labelledreagent, and blocking agents to reduce background signal caused bynon-specific binding of the labelled reagent, which blocking agents mayinclude serum, bovine serum albumin or gelatin.

In addition, since the peptide of SEQ ID NO 1 has been identified as acytotoxic element in C. albicans infections, it can be used to developdrugs and other therapies that specifically target this peptide. It maytherefore form a useful tool in the screening of antifungal compounds.Screening methods of this type form a further aspect of the invention.

Screening methods may take a variety of forms. Typically however,compounds under test will be contacted with a peptide of the inventionand in particular a peptide of SEQ ID NO 1 and any interaction betweenthe peptide and the compound will be monitored. The interaction may takeplace in a vessel or well for instance of a 96-well plate, and theinteraction detected using for example, a change in reflectance ofpolarised light caused by binding. Alternatively, the interaction maytake place at a surface at which one of the peptide or the targetreagent is immobilised and under conditions where interaction may bedetected using techniques such as surface plasmon resonance and thelike.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be particularly described by way of example withreference to the accompanying diagrams which are summarised as follows.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the invention. Thefollowing descriptions of specific embodiments of the present inventionare presented for purposes of illustration and description. They are notintended to be exhaustive of or to limit the invention to the preciseforms disclosed. Many modifications and variations are possible in viewof the above teachings. The embodiments are shown and described in orderto best explain the principles of the invention and its practicalapplications, to thereby enable others skilled in the art to bestutilize the invention and various embodiments with various modificationsas are suited to the particular use contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C is a series of graph showing the results of an experiment todetermine whether ECE1 is required for damage of a variety of hostcells. Oral TR146 (FIG. 1A), gastrointestinal Caco (FIG. 1B) and vaginal(FIG. 1C) epithelial cells were infected with C. albicans wild type(wt), ece1Δ or ece1Δ/Δ-ECE1 strains for 24 hours and damage assayed byLDH release as described below.

FIG. 2 shows the structure of Ece1 and phylogeny with other species. Itshows the alignment of Ece1 from C. albicans (SEQ ID NO 13) andorthologues in C. dubliniensis (SEQ ID NO 14) and C. tropicalis (SEQ IDNO 15) (which are the only three sequenced fungal species that encodeEce1). The bars below the alignment indicate that the peptides resultedfrom Kex2 digestion of the CaEce1. Only peptide 3 of C. albicans and C.dubliniensis form transmembrane α-helical structures.

FIG. 3A-C is a series of graphs showing the results of an experiment todemonstrate that a peptide of the invention is sufficient to lyse hostepithelial cells. Mixtures of Ece1 peptides at different concentrationsor the peptide of SEQ ID NO 1 alone were added to TR146 oral epithelialcells in the presence of the ece1Δ mutant, incubated for 24 hours anddamage assessed by measuring LDH release (FIG. 3A). Subsequently thedifferent Ece1 peptides were added alone (without C. albicans cells) toTR146 (FIG. 3B) and vaginal A431 (FIG. 3C) epithelia, incubated for 24hours and damage assessed by measuring LDH release as described below.

FIG. 4 is a graph showing the results of an experiment to determinewhich of the Ece1 peptides are haemolytic toxins where the peptide ofSEQ ID NO 1 is peptide 3. Individual peptides were incubated with humanerythrocytes for 1 hour and heamolysis assayed by measuring haemoglobinrelease (as measured by absorbance at 541 nm, normalised to a 100% lysiscontrol).

FIG. 5A,B shows the results obtained by scanning electron microscopy ofan experiment to determine whether the peptide of SEQ ID NO 1 formspores in epithelial membranes. Oral epithelial (TR146) monolayers wereincubated with 5 μg/ml peptide 3 (SEQ ID NO 1) for 5 hours and poreformation assessed by scanning electron microscopy. FIG. 5B shows afurther magnified view of a region indicated in FIG. 5A.

FIG. 6A,B is a series of graphs showing the results of an experiment todemonstrate that the haemolytic activity of the Ece1 peptide 3 of SEQ IDNO 1 is ion-dependent. The peptide was incubated with human erythrocyteswith increasing concentrations of the divalent cation chelator EDTA(FIG. 6A) and haemolysis assayed by measuring haemoglobin release(absorbance at 541 nm, normalised to a 100% lysis control). Theinhibitory effect of 50 mM EDTA was reversed by the addition of 50 or100 mM magnesium (FIG. 6B).

FIG. 7A,B is a series of graphs showing the results of experiments toinvestigate whether the peptide of the invention activates epithelialimmunity and cFos. (FIG. 7A) Induction of c-Fos DNA binding activity inTR146 epithelial cells after 3 hours infection with ece1Δ/Δ null mutant,ece1Δ/Δ-ECE1 revertant and the BWP17 parent strain. (FIG. 7B) Productionof cytokines by TR146 epithelial cells after 24 hour infection withece1Δ/Δ null mutant, ece1Δ/Δ-ECE1 revertant, the BWP17 parent strain oruninfected cells. Data shown are the mean of 3 independent experiments.Error bars show SEM. *=p<0.05, **=p<0.01, ***=p<0.001.

FIG. 8A,B shows the amino acid sequences of and WTSA peptides where FIG.8A shows the amino acid sequence of (SEQ ID NO 1). Amyloidogenic regionsare shown in bold type; and FIG. 8B shows the amino acid sequences ofWTSA peptides. Each individual WTSA peptide constitutes a partialfragment of full length Ece1-III. Amyloidogenic regions are shown inbold type.

FIG. 9 is a graph showing the results of analysis of WTSApeptide-induced damage to TR146 oral epithelial cells. Individual WTSApeptides (250 μg/ml) and WTSA 3 in combination with WTSA 6 (bothpeptides at 250 μg/ml) were added to confluent monolayers of TR146 oralepithelial cells and incubated for 24 hours at 37° C., 5% CO₂. Followingincubation, growth medium was removed and assayed for the presence oflactate dehydrogenase (LDH), a surrogate marker of cellular lysis.Dimethyl sulfoxide (DMSO) was used as a vehicle only (negative control).Full length Ece1-III (250 μg/mL) was used as the positive control.

FIG. 10A-E shows WTSA peptide-mediated activation of immune responses inTR146 oral epithelial cells. WTSA peptides 1-6 were added to confluentmonolayers of TR146 oral epithelial cells at a final concentration of250 μg/mL and incubated for 24 hours at 37° C., 5% CO₂. Followingincubation, growth medium was removed and secreted immunostimulatorycytokines were quantified by multiplex bead assay. Concentration ofdetected cytokines was expressed as picograms per millilitre (pg/mL).ND=not detected.

FIG. 11A-C is a series of graphs showing the results of experiments toinvestigate whether the peptide of the invention binds EGFR andactivates c-Fos. (FIG. 11A) Induction of c-Fos DNA binding activity inTR146 epithelial cells after 3 hours infection with wild-type C.albicans cells, which were pre-treated for 1 hour with 4 μM GW2974(EGFR/Her2 inhibitor), 1 μM PD153035 (EGFR inhibitor) or DMSO (vehiclecontrol). (FIG. 11B) Sensorgram of fluid phase EGFR binding toimmobilised Ece1-III of SEQ ID NO 1 demonstrating high affinity binding.Injection of fluid phase components was at point A. (FIG. 11C) Inductionof c-Fos DNA binding activity in TR146 epithelial cells after 3 hoursinfection with Ece1-III, which were pre-treated for 1 hour with 4 μMGW2974 (EGRF/Her2 inhibitor), 1 μM PD153035 (EGFR inhibitor) or DMSO(vehicle control). Data shown are the mean of 2(A) or 3 (C) independentexperiments. Error bars show SEM. *=p<0.05, **=p<0.01, ***=p<0.001.

FIG. 12A-C is a set of graphs showing the effect of EGFR inhibition onepithelial cell signalling. Blocking EGFR signalling in oral epithelialcells (TR146) with Gefitnib (10 μM) inhibits the activation of MAPKintracellular signalling in cells treated with Ece1-III (50 μg/mL) for 2hours as measured by decreases in levels of phosphorylated p38, JNK andERK1/2 relative to the vehicle control (DMSO).

FIG. 13 shows the results of an assay to detect Ece1-III-phospholipidinteractions. Ece1-III was used to probe a phospholipid array (PIP Stripmembrane). Binding was assessed by hybridisation with anti-Ece1-IIIantibody, followed by detection with anti-rabbit antibody conjugated tohorse radish peroxidase.

FIG. 14A-C is a series of graphs showing the results of an experiment todetermine C. albicans-macrophage interactions. C. albicans strains wereincubated with RAW264.7 macrophages for indicated times and thefollowing parameters determined: (FIG. 14A) The number of phagocytosedfungal cells as a percentage of total number of fungal cells; (FIG. 14B)Hyphal length; (FIG. 14C) The percentage of outgrowing hyphae comparedto the total number of internalised yeast mother cells.

FIG. 15 is a graph showing the results of an experiment to show thatECE1 is required for damage of macrophages. Indicated macrophage celltypes were co-incubated with C. albicans cells for 24 hours (MOI of 1for Raws, MOI of 10 for MDMs) and macrophage damage assessed bymeasuring lactate dehydrogenase release into the supernatant.

FIG. 16A-E shows the results of experiments showing the role of Ece1 inC. albicans mucosal pathogenesis. Infection of cortisone acetate-treatedmice with wild-type (BWP17), ece1Δ null mutant or ece1Δ/Δ-ECE1 revertedstrain (FIG. 16A) numbers of C. albicans fungi recovered per gram ofkidney 3 days post-infection. Fungal invasion, tissue damage andinflammatory cell infiltration of tongue tissue 3 days post-infection by(FIG. 16B) BWP17, (FIG. 16C) ece1Δ/Δ-ECE1 and (FIG. 16D) ece1Δ nullmutant, indicating that the ece1Δ null mutant does not invade, damage orrecruit inflammatory cells. (FIG. 16E) tongue section 3 dayspost-infection with BWP17 immunostained with MPO and CD15 markers ofneutrophils. **=P<0.01.

FIG. 17 is a graph showing the results of an experiment to showDrosophila killing by Ece1-III. The percentage of Drosophila death, 24hours post-infection of 250 μg/ml His-tagged full length Ece1p, Ece1-II(peptide 2) (FIG. 2), and Ece1-III (peptide 3), into a cohort of 20age-matched flies.

EXAMPLE 1 The Role of Ece1 in Host Cell Damage

To investigate the role of Ece1 in host cell damage, a series of C.albicans gene deletion mutants lacking ECE1, as well as revertant mutantstrains were created. Specifically, C. albicans gene deletion cassetteswere generated using the PCR-based method described by Dalle F et al.Cellular Microbiology 2010; 12:248-71. Primers ECE1-FG and ECE1-RG asshown below as SEQ ID NO 4 and 5 respectively, were used to amplify theHIS1 and ARG4 markers from plasmids pFA-HIS1 and pFA-ARG4, respectively.The C. albicans strain BWP17 (Wachtler B, et al. Antimicrobial Agentsand Chemotherapy 2011; 55:4436-9) was sequentially transformed asdescribed by Wachtler B, et al. PloS one 2011; 6:e17046, with theECE1-ARG4 and ECE1-HIS1 deletion cassettes and then transformed with theCandida integrating plasmid 10 or CIp10 (Murad et al. Yeast (2000) 16,4, p 325-327), yielding the ece1Δ deletion strain. For generation of thecomplemented strain, the ECE1 open reading frame, plus upstream anddownstream intergenic regions were amplified with primers ECE1-RecF3k(SEQ ID NO 6) and ECE1-RecR (SEQ ID NO 7) and cloned into plasmid CIp10at MluI and SalI sites, yielding plasmid CIp10-ECE1. This plasmid wastransformed into the uridine auxotrophic ece1Δ-deletion strain, yieldingthe ece1Δ/Δ-ECE1 complemented strain.

Primers:

ECE1-FG (SEQ ID NO 4) ATCAAATAACCCACCTATTTCAAAATTGTTTTATTTTTGTTTATCTCTACAACAAACAACTTTCCTTTATTTTACTACCAACTATTTTCCATTCGTTAAAgaagcttcgtacgctgcaggtc ECE1-RG (SEQ ID NO 5)CACAAAAAACAACAATTAAAAAAATCAGTTACAGCAAAAGTGTCACAAGACTTATGGAATAAAAGATTAAGCTTGTGGAAAACAAATTTTTATCTGCTGAGCATtctgatatcatcgatgaattcgag ECE1-RecF3k (SEQ ID NO 6)GCACGCGTCTAAAGTGGAGTAACAAC ECE1-RecR (SEQ ID NO 7)GGTCGACCCCAGACGTTGGTTGC

Three strains, wild type C. albicans (wt), the ECE1 deletion mutant(ece1Δ) and the revertant mutant strain (ece1Δ/Δ-ECE1) were tested. Theywere applied to three independent human epithelial cell types: oral,gastrointestinal and vaginal. TR146, Caco-2 and A431 epithelial celllines were maintained as previously described in the Dalle and Wachtlerreferences given above. Epithelial damage assays were performed aspreviously described by Dalle et al. (supra.) and Wachtler et al. (2011)(supra.) with the following modifications: monolayers in 96 well plateswere infected with 2×10⁴ C. albicans cells; infections were performed incell culture media without fetal bovine serum.

After incubation for 24 hours, the damage to the cells was assessed bymeasuring lactate dehydrogenase (LDH) release using a conventional assaymethod.

The results are shown in FIG. 1A-C. They show that deletion of ECE1,prevented or reduced C. albicans damage in all three cell types. As C.albicans is often found associated with the oral, gastrointestinal andurogenitary sites as a member of the natural microbiome, but can alsocause infections at such sites, these data suggest that the ECE1 geneproduct is involved C. albicans pathogenicity.

EXAMPLE 2 Role of Peptides

It has been demonstrated that Ece1 can be proteolytically processed byKex2 into eight peptide fragments (O. Bader et al. BMC Microbiology2008, 8:115). FIG. 2 illustrates the structure of Ece1 and the proposedresultant peptide fragments following proteolytic processing as well asits phylogenic relationship with orthologues in C. dubliniensis and C.tropicalis (note that ECE1 orthologues are only found in these twofungal species). Each of the individual C. albicans peptides weresynthesised by ProteoGenix using fmoc solid phase peptide synthesistechnology.

The method of Example 1 was then repeated using a 1:1 mixture ofpeptides (1-7) of FIG. 2 on human TR146 oral epithelial cells at variousconcentrations (250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, 15.625μg/ml and 7.8 μg/ml of each individual peptide within the mixture) orpeptide 3 (SEQ ID NO 1) alone in the presence of the ece1Δ mutant C.albicans. Peptide 8 could not be included, as this peptide wasinsoluble. After incubations for 24 hours, the damage was assessed bymeasuring LDH release as before. The results are shown in FIG. 3A. Thisshows that peptide 3 (designated Ece1-III in the Figure) producedsimilar levels of damage to the combination at a concentration of 250μg/ml.

The test was then repeated using each peptide individually without C.albicans cells. The results are shown in FIGS. 3B and 3C. They show thatwhilst peptide 3 was capable of damaging both oral epithelial andvaginal cells, the other peptides displayed essentially no cytolyticactivity. Therefore, peptide 3 of processed Ece1 (“Ece1-III” of SEQ IDNO 1) is sufficient to cause lysis of human epithelial cells.

EXAMPLE 3 Cytolytic Activity of Peptides

In order to investigate the cytolytic activities of Ece1-III in greaterdetail, a haemolysis assay was carried out. Human blood was collectedwith the SARSTEDT blood collection system and EDTA-coated tubes, washedonce with HBSS, resuspended in HBSS and stored at 4° C. for up to oneweek for experiments. Blood was obtained from healthy human donors.

Each individual peptide (at a final concentration of 30 μg/ml) wasincubated with 1×10⁷ human erythrocytes in a volume of 150 μl. Afterincubation for 1 hour at 37° C., haemolysis was assayed by measuringhaemoglobin release, as indicated by absorbance at 541 nm, normalised toa 100% lysis-water control.

The results are shown in FIG. 4. Exposure of human erythrocytes toEce1-III, but not to the other peptides, resulted in lysis of thesecells. It should be noted that, in contrast to epithelial cells,erythrocytes do not contain nuclei and cannot transcriptionally respondto stimuli. Therefore, lysis of erythrocytes by Ece1-III was due todirect cytolysis.

EXAMPLE 4 Pore Formation

Many peptide toxins elicit cytotoxicity via pore formation and ioninflux across membranes. Whether addition of Ece1-III to epithelialcells was able to directly induce pore formation was then tested. Oralepithelial (TR-146) monolayers were incubated with 5 μg/ml Ece1-III ofSEQ ID NO 1 for 5 hours and pore formation assessed by scanning electronmicroscopy. The results are shown in FIG. 5A,B where FIG. 5B shows afurther magnified view of the region shown in FIG. 5A. Electronmicroscopy suggests that Ece1-III can directly induce pores (FIG. 5A,B), which is in agreement with the view that Ece1-III can act as apore-forming toxin.

EXAMPLE 5 Ion-Dependency of Haemolytic Activity

Since the mode of action of many peptide and pore-forming toxins ision-dependent, Ece1-III and erythrocytes were co-incubated in thepresence of increasing concentrations of the divalent cation chelator,ethylenediaminetetraacetic acid (EDTA), which restricts thebioavailability of divalent cations. After 1 hour, haemolysis wasmeasured as described in Example 3 above. The results, shown in FIG. 6A,indicate that increasing concentrations of EDTA blocked the haemolyticactivity of Ece1-III. This effect was reversed by supplementation withmagnesium (FIG. 6B). Therefore, like previously described peptidetoxins, the haemolytic activity of Ece1-III appears to be ion-dependent.

EXAMPLE 6 Activation of Immune Responses

Pore-forming toxins are generally strong activators of inflammatoryresponses. Previously it has been shown that recognition of C. albicanshyphae resulted in the activation of the mitogen-activated proteinkinase (MAPK) p38 signalling pathway and the c-Fos transcription factor,which in turn activates proinflammatory cytokine production (Moyes D L,et al.: Cell Host Microbe 2010, 8:225-235,). An extensive screen of >100C. albicans mutants was carried out using protocols described in thisreference and also Moyes D L, et al. PLoS ONE 2011, 6:e26580; MurcianoC. et al. Infect. Immun 2011, 79:4902-4911; Moyes D L, et al. MedMicrobiol Immunol 2012, 201:93-101; Moyes D L, et al. Methods Mol Biol2012, 845:345-360 and Murciano C, et al. PLoS ONE 2012, 7:e33362.

The results demonstrated that only a strain deleted in ECE1 was unableto activate the c-Fos pathway (FIG. 7A) or proinflammatory cytokines(FIG. 7B) from oral epithelial cells whilst still producing hyphae.Therefore, activation of epithelial immune responses via c-Fos againstC. albicans is the result of the action and/or recognition of Ece1.

EXAMPLE 7 Functional Requirements of Ece1-III

Having identified Ece1-III as the region of Ece1 responsible forcellular lysis and the activation of immune responses, the applicantsnext investigated the precise functional requirements of the Ece1-IIIamino acid sequence (FIG. 8A). Accordingly, individual peptide fragmentscorresponding to internal regions of Ece1-III were constructed (WTSApeptides 1-6 (SEQ ID NOS 8-11, 3 and 12 respectively: FIG. 8B). Thesequence of each WTSA peptide was chosen to facilitate a detailedexamination of each of the internal amyloidogenic regions of Ece1-III,either alone (WTSA peptides 1, 2, 4 and 5), or in combination (WTSA 3).An additional fragment of Ece1-III lacking both amyloidogenic regionswas also created (WTSA 6: FIG. 8B). The WTSA peptides were analysed fortheir ability to cause epithelial cell lysis. However, none of theindividual WTSA peptide fragments were observed to induce cell lysis(FIG. 9). Notably, when WTSA 3 and 6 were applied to epithelial cells incombination, (which together constitute the entire Ece1-III peptidesequence) the ability to induce cell lysis was not restored. The WTSApeptides were then analysed for their ability to stimulate secretion ofpro-inflammatory cytokines from oral epithelial cells. None of the WTSApeptides stimulated significant levels of cytokine secretion (FIG.10A-E). In contrast, a potent induction of all cytokines was observedfollowing exposure to full length Ece1-III. Taken together, these dataindicate that only intact, full length Ece1-III is capable of inducingcellular lysis and stimulation of the inflammatory response inepithelial cells.

The p38/c-Fos pathway is activated via the interaction of C. albicanshyphae with the surface receptor EGFR (epidermal growth factorreceptor), as blocking EGFR activation also blocks c-Fos activation(FIG. 11A). Biacore binding assays show that Ece1-III (of SEQ ID NO 1),but not other parts of the Ece1 protein, directly interact with EGFRwith high affinity (FIG. 11B) and that blocking EGFR abolishes theability of Ece1-III to activate c-Fos (FIG. 11C). Furthermore, blockingEGFR also reduced/abolished activation of all three MAPK signallingpathways (p38, JNK and ERK1/2) (FIG. 12A-C) This demonstrates thatactivation of mucosal immune responses via the p38/c-Fos pathway is theresult of Ece1-III/EGFR interactions and that EGFR plays a primary rolein the activation of cell signalling by Ece1-III.

EXAMPLE 8

Interaction of Ece1-III with Phospholipids

In order to insert into a target membrane, a pore-forming peptide toxinmust interact with components of the membrane. Because Ece1-III consistsof a predicted alpha helix with a dibasic (lysine, arginine) C-terminalhead, the applicants predicted that these cationic, positively chargedresidues may interact with negatively charged phospholipids present intarget membranes. Ece1-III was used to probe a phospholipid array (PIPStrip membrane). Binding was assessed by hybridisation withanti-Ece1-III antibody, followed by detection with anti-rabbit antibodyconjugated to horse radish peroxidase. The results are shown in FIG. 13.These show that Ece1-III interacts withPhosphatidylinositol-3-phosphate, Phosphatidylinositol-4-phosphate,Phosphatidylinositol-5-phosphate, Phosphatidylinositol-3,4-phosphate,Phosphatidylinositol-3,5-phosphate, Phosphatidylinositol-4,5-phosphate,Phosphatidylinositol-3,4,5-phosphate, Phosphatidic acid andPhosphatidylserine, with binding to Phosphatidylinositol-3-phosphatebeing particularly robust (FIG. 13). Ece1-III did not interact withLysophosphatidic acid, Lysophosphatidylcholine, Phosphatidylinositol,Phosphatidylethanolamine, Phosphatidylcholine orSphingosine-1-phosphate. Therefore, insertion of Ece1-III into targetmembranes (pore formation) may be mediated by interactions withphospholipids.

EXAMPLE 9 Ece1 Facilitates Immune Cell Killing

Given the importance of Ece1-III in killing epithelial cells, theapplicants examined whether it also plays a role in immune escape of C.albicans from macrophages. In vitro, C. albicans yeast cells arephagocytosed by macrophages, but rapidly germinate and escape themacrophage via hypha formation. To examine whether Ece1 is involved inthis process, wild type, ece1Δ, and ece1Δ/Δ-ECE1 cells were coincubatedwith macrophages and the following parameters measured: phagocytosisrates, length of hyphae, escape from macrophages, and damage ofmacrophages. Interestingly, ece1Δ cells were phagocytosed by macrophagesat the same rate, formed hyphae within macrophages of the same length,and even escaped from macrophages at the same rates as the wild type,but were unable to damage these immune cells (FIGS. 14A-C and 15). Thesedata demonstrate that C. albicans hyphae can non-lytically escape frommacrophages, and indicate a specific role for Ece1 in host cell damage.

EXAMPLE 10

Effect of Ece1 on Mucosal pathogenicity In Vivo

The applicants also tested whether Ece1 was required for mucosalpathogenciity in vivo. To test this, they utilised a murine model oforopharyngeal candidiasis. In this model, mice were firstimmunosuppressed with cortisone acetate prior to administration ofwild-type (parental) C. albicans (BWP17), a ece1Δ null mutant andece1Δ/Δ-ECE1 revertant strain into the oral cavity. After four days,fungal burdens, fungal invasion, tissue damage and immune cellrecruitment was determined in tongue tissues. Significantly lower fungalburdens were found in mice infected with the ece1Δ null mutant ascompared with the wild-type (BWP17) or the ece1Δ/Δ-ECE1 revertantstrains, and in many cases the ece1Δ null mutant could not be recoveredfrom mice (FIG. 16A). Histological analysis of mouse tongues in both thewild-type (BWP17) and ece1Δ/Δ-ECE1 revertant strain showed multiple fociof infection that were associated with inflammatory immune cells(neutrophils) and extensive local tissue damage (FIG. 16B-D). Incontrast, mice infected with the ece1Δ null mutant showed no evidence ofany foci of invasion, tissue damage or inflammation and no evidence ofC. albicans was detectable (FIG. 16E). Therefore, Ece1 is essential forsuccessful mucosal infection, damage induction and immune activation inthis model.

EXAMPLE 11

Effect of Ece1 and Ece1-III on Drosophila melanogaster

The applicants also determined the effect of administering full lengthHis-tagged Ece1p and Ece1-III in a Drosophila melanogaster (fruit fly)model. All flies injected with full length His-tagged Ece1p and Ece1-IIIdisplayed signs of paralysis, with only 17.5% and 10% recovering after24 hours, as compared with the negative control Ece1-II (peptide 2)which showed no signs of paralysis (FIG. 17). This indicates thatEce1-III may have a direct or indirect neurotoxic effect.

CONCLUSION

These data provide evidence that Ece1 (and its active componentEce1-III) has a dual functional role, by acting both as a pore-formingtoxin that damages host cells and as an activator of immune responses.Damage of host cells and uncontrolled immune activation are thehallmarks of several diseases caused by C. albicans, which can oftenlead to death (45-76% mortality) during systemic infections. Therefore,neutralisation of Ece1-III will prevent not only host damage during C.albicans infections but also deleterious inflammatory responses. Thisindicates that Ece1-III represents a new therapeutic target to treat C.albicans infections.

1. A specific binding partner for a peptide comprisingSIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR (SEQ ID NO:1) or a labelled formthereof.
 2. The specific binding partner of claim 1 which is animmunoglobulin.
 3. The specific binding partner of claim 2 which is anantibody or a binding fragment thereof.
 4. The specific binding partnerof claim 3 which is a monoclonal antibody.
 5. A pharmaceuticalcomposition comprising the specific binding partner of claim 1 and apharmaceutically acceptable carrier.
 6. A method for prophylaxis ortreatment of an infection by Candida species, said method comprisingadministering to a subject in need thereof an effective amount of thespecific binding partner of claim
 1. 7. The method of claim 6 whereinthe infection is an oral or mucosal infection by Candida albicans.
 8. Anucleic acid that encodes the specific binding partner of claim
 1. 9. Arecombinant cell that has been transformed with the nucleic acid ofclaim
 8. 10. A method for producing the specific binding partner encodedby the nucleic acid that transformed the recombinant cell of claim 9,which method comprises culturing the recombinant cell and recoveringsaid specific binding partner.
 11. A method for diagnosis of aninfection by Candida albicans, said method comprising contacting asample from an individual with the specific binding partner of claim 1,and detecting whether the sample contains moieties that interact withsaid specific binding partner at a sufficient level to indicate apathogenic invasive infection.
 12. A kit for diagnosing an infection byCandida albicans, said kit comprising the specific binding partner ofclaim 1, and means for detecting complexes formed with said specificbinding partner.
 13. A kit for diagnosing an infection by Candidaalbicans, said kit comprising a peptide comprisingSIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR (SEQ ID NO:1), and means for detectingcomplexes formed with said peptide
 14. A method for diagnosis of aninfection by Candida albicans, said method comprising contacting asample from an individual with the peptide from the kit of claim 13, anddetecting whether the sample contains moieties that interact with saidpeptide at a sufficient level to indicate a pathogenic invasiveinfection.
 15. A method for screening for compounds having anti-fungalactivity, said method comprising determining whether a test compoundwill interact with a peptide comprising SIIGIIMGILGNIPQVIQIIMSIVKAFKGNKR(SEQ ID NO:1).